Alveoli mediate gas exchange and are thus the key functional structure of the lung, and they are also the site of many important and intractable lung diseases. While its complex grapelike structure is comprised of several cell types (e.g., epithelial, mesenchymal, endothelial, and hematopoietic), the alveolar epithelium itself is quite simple and contains only two alveolar cell types (AT); the exquisitely thin AT1 cells that form the site of gas exchange and the cuboidal AT2 cells which secrete the surfactant that prevents alveolar collapse. We recently described the cellular process by which these cell types arise: a bipotent progenitor (BP) gives rise to both AT1 and AT2 fates. We also recently conducted single cell gene expression profiling of the developing alveolar epithelium, and used the data to reconstruct the full gene expression program of the lineage hierarchy from BP to both AT1 and AT2 fate. However, the signals that control this program are unknown. I propose to gene expression program of the developing the alveolar epithelial lineage hierarchy with expression profiling of surrounding alveolar cells to identify a critical receptor and its cognate ligand combine the that induce alveolar epithelial development. Understanding how AT fate specification is determined during development will provide significant insight to how it is disrupted in diseases like bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and lung adenocarcinoma as well as help in devising regenerative strategies to repair or replace diseased tissue. The mentored phase of the proposal will be carried out at Stanford University in Dr. Mark Krasnow's laboratory, a leading lab for training developmental and stem cell biologists. The mentored phase of the proposal focuses on identifying a signaling pathway that controls alveolar development. Towards this goal, I will 1) Use single RNAseq expression profiling to identify all receptor genes selectively expressed in the developing AT1 and AT2 lineage and to identify expression in surrounding alveolar mesenchymal and endothelial cells of any genes that encode their cognate ligands. This will identify outstanding candidates for the signaling pathways that control alveolar epithelial fate specification. For the second aim, I will 2) Elucidate the role of one candidate signaling pathway, the Fgf7-Fgfr2 pathway identified by initial expression profiling results, in alveolar development, by determining if the pathway is necessary and sufficient for alveolar epithelial fate specification, and what alveolar cells the pathway is required in to exert its effet. Preliminary data show that purified Fgf7 is a powerful inducer of alveolar cell development and alveolar morphogenesis in a newly established cell culture assay, so it provides a promising test case for elucidating the role of an identified candidate signaling pathway in alveolar development. If successful, I will have identified the first important signaling pathway for alveolus development, and a framework for identifying and characterizing the other important signals. This will provide critical information on lung development and a rational approach to toward regenerative alveolar therapies. From Dr. Krasnow's laboratory, I will pursue my goal of finding a tenure track position to complete the the independent phase of the proposal. This phase focuses on characterizing how the alveolar microenvironment is constructed during development, a poorly understood aspect of alveolar development that is critical for understanding alveolar morphogenesis and ultimately bioengineering an alveolus. Toward this goal, I will 3) Determine the composition and pattern of the alveolar microenvironment during alveologenesis. Composition will be determined by analysis of single cell transcriptional profiles of alveolar epithelial, mesenchymal and endothelial cells for expression of extracellular matrix (ECM) and ECM-related genes, and then mapping the spatial distribution of the ECM proteins during alveolar development, to construct a 4-D map depicting their relationship to each other and each of the major cell types and how this changes during alveolar development. My long term goal is to identify and characterize the cells, control signals and microenvironment of the developing alveolus to guide tissue engineering of a healthy alveolus. I believe this proposal is innovative in three ways. First, it seeks to identify and characterize the first molecular control signals that induce and guide alveolar development. Second, it develops a systematic in vitro and in vivo approach to guide future studies identifying and characterizing other important alveolar development signals. Finally, the independent phase is the first attempt to systematically characterize the formation of the much less studied but equally important component of the alveolus; the microenvironment. From these maps of alveolar development I hope one day to be able to engineer a synthetic alveolar microenvironment and then seed it with isolated progenitors/stem cells and add purified signals to create a functional alveolus for tissue replacement therapies.